Genome-Wide Analysis of Light- and Temperature- Entrained...

Genome-Wide Analysis of Light- and Temperature-Entrained Circadian Transcripts in CaenorhabditiselegansAlexander M. van der Linden1*¤a, Matthew Beverly1, Sebastian Kadener1¤b, Joseph Rodriguez1,2, Sara

Wasserman1, Michael Rosbash1,2, Piali Sengupta1*

1 Department of Biology and National Center for Behavioral Genomics, Brandeis University, Waltham, Massachusetts, United States of America, 2 Howard Hughes Medical

Institute, Brandeis University, Waltham, Massachusetts, United States of America

Abstract

Most organisms have an endogenous circadian clock that is synchronized to environmental signals such as light andtemperature. Although circadian rhythms have been described in the nematode Caenorhabditis elegans at the behaviorallevel, these rhythms appear to be relatively non-robust. Moreover, in contrast to other animal models, no circadiantranscriptional rhythms have been identified. Thus, whether this organism contains a bona fide circadian clock remains anopen question. Here we use genome-wide expression profiling experiments to identify light- and temperature-entrainedoscillating transcripts in C. elegans. These transcripts exhibit rhythmic expression with temperature-compensated 24-hperiods. In addition, their expression is sustained under constant conditions, suggesting that they are under circadianregulation. Light and temperature cycles strongly drive gene expression and appear to entrain largely nonoverlapping genesets. We show that mutations in a cyclic nucleotide-gated channel required for sensory transduction abolish both light- andtemperature-entrained gene expression, implying that environmental cues act cell nonautonomously to entrain circadianrhythms. Together, these findings demonstrate circadian-regulated transcriptional rhythms in C. elegans and suggest thatfurther analyses in this organism will provide new information about the evolution and function of this biological clock.

Citation: van der Linden AM, Beverly M, Kadener S, Rodriguez J, Wasserman S, et al. (2010) Genome-Wide Analysis of Light- and Temperature-Entrained CircadianTranscripts in Caenorhabditis elegans. PLoS Biol 8(10): e1000503. doi:10.1371/journal.pbio.1000503

Academic Editor: Ueli Schibler, University of Geneva, Switzerland

Received June 7, 2010; Accepted August 19, 2010; Published October 12, 2010

Copyright: � 2010 van der Linden et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permitsunrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: This work was supported by the National Institutes of Health (RO1 GM081639 to PS; P30 NS045713, PO1 044232 to MR; F31 NS060584 to SW), aNational Science Foundation IGERT training grant (DGE-0549390 to MB and JPR), a Human Frontiers Science Program Long-term Fellowship and CareerDevelopment Award (SK), and the Howard Hughes Medical Institute (MR). The funders had no role in study design, data collection and analysis, decision topublish, or preparation of the manuscript.

Competing Interests: The authors have declared that no competing interests exist.

Abbreviations: CC, constant cold; CW, cold/warm; DD, constant darkness; FUDR, 5-fluoro-2-deoxyuridine; gfp, green fluorescent protein; GO, Gene Ontology; LD,light/dark; QQ, quantile–quantile; qRT-PCR, quantitative reverse transcription PCR; s.e.m., standard error of the mean; WC, warm/cold

* E-mail: [email protected] (AMvdL); [email protected] (PS)

¤a Current address: Department of Biology, University of Nevada, Reno, Nevada, United States of America¤b Current address: Department of Biological Chemistry, Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem, Israel

Introduction

Organisms exhibit daily (circadian) rhythms in behavior and

physiology that are attuned to the earth’s rotational period. The

periods of these intrinsic rhythms are approximately 24 h and are

synchronized by external cues or zeitgebers (time givers) such as

light and temperature. The molecular composition and regulation

of endogenous pacemakers that generate circadian rhythms have

been extensively investigated. Both the molecules composing the

core clock and the molecular mechanisms of clock function are

remarkably divergent across kingdoms, as well as between

eukaryotes and prokaryotes, suggesting multiple, independent

evolutionary origins for the circadian clock [1–6]. However, genes

and pathways by which circadian oscillatory rhythms are

generated in animals such as Drosophila and mammals are strongly

conserved [5,7,8], implying a single origin of the clock in the

metazoan lineage [2]. Thus, it is of interest to identify and

characterize the circadian clock in additional animals.

The nematode Caenorhabditis elegans is well established as an

excellent model organism for the study of development and

behavior [9–12]. In their soil habitat, worms are subjected to

temperature fluctuations corresponding to solar irradiance during

the day–night cycles [13,14]. Moreover, when associated with

surface-dwelling animals [15], C. elegans is likely to be exposed to

daily light–dark cycles. Several studies have previously reported the

presence of circadian rhythms in C. elegans. In these studies, growth-

synchronized populations were entrained to light or temperature

cycles, and circadian rhythms in behaviors such as locomotion or

responses to osmotic stress were quantified [16–20]. Although these

experiments demonstrated the presence of rhythms with character-

istics of true circadian oscillations, these rhythms were surprisingly

variable and non-robust [19]. In addition, while the C. elegans

genome is predicted to encode homologs of most Drosophila and

mammalian core clock components, the roles of these genes appear

to be largely restricted to development [21]. Thus, whether C. elegans

exhibits bona fide circadian rhythms remains unclear.

PLoS Biology | www.plosbiology.org 1 October 2010 | Volume 8 | Issue 10 | e1000503

Circadian rhythmicity in behavior and physiology has been

proposed to be driven by clock-regulated oscillations in transcrip-

tion and translation, and/or via post-transcriptional or post-

translational modifications [22–32]. The expression of core clock

genes such as per (period) and tim (timeless) as well as many clock

output genes cycles at the transcriptional level in a circadian

manner in Drosophila [23,24]. Expression profiling experiments

have, therefore, provided a powerful approach to identify clock

genes as well as clock output genes under circadian transcriptional

control [33–41]. Since expression profiling is not biased by prior

assumptions regarding a specific behavioral or physiological

output, transcriptional rhythms may be a robust measure of the

presence of a molecular circadian clock.

Here we identify light- and temperature-regulated transcrip-

tional rhythms in C. elegans and show that subsets of these

transcripts are regulated in a circadian manner. We find that light

and temperature also globally drive the expression of many genes,

indicating that C. elegans exhibits systemic responses to these

stimuli. Light- and temperature-entrained transcripts appear to be

largely nonoverlapping. We show that conserved clock gene

homologs do not exhibit circadian rhythmicity at the mRNA level.

We also find that the TAX-2 cyclic nucleotide-gated channel

subunit, previously implicated in thermosensory signal transduc-

tion and phototransduction [42–44] is required to transduce both

light and temperature information to the clock(s). These results

demonstrate that C. elegans has circadian clock(s) that are entrained

by light and temperature, and indicate that this model organism

can be used to further explore the evolution and function of this

critical timekeeping mechanism.

Results

Identification of Cycling Transcripts Entrained by Lightand Temperature

To determine whether cycling transcripts can be identified in C.

elegans following entrainment to either light–dark or temperature

cycles, we performed a genome-wide expression profiling analysis.

Growth-synchronized populations of wild-type L1 larvae were

entrained until adulthood to either 12 h:12 h light/dark (LD)

cycles at a constant temperature of 18uC, or to 25uC:15uC (warm/

cold [WC]) temperature cycles in constant darkness (DD)

(Figure 1A). Following entrainment, animals were maintained

under free-running conditions of DD or at a constant temperature

of 15uC (constant cold [CC]) (Figure 1A). To exclude non-

entrained transcripts present in the embryos of self-fertilizing C.

elegans hermaphrodites, L4 larvae and adults were grown on plates

containing 5-fluoro-2-deoxyuridine (FUDR), which inhibits DNA

synthesis and results in embryonic lethality [45]. RNA was

collected every 4 h during the last 24 h of entrainment as well as

during the first 24 h of free-running conditions (Figure 1A) and

hybridized to Affymetrix C. elegans Genome Arrays containing

22,500 probe sets representing more than 19,000 predicted C.

elegans genes.

Figure 1. Method for RNA collection and analysis flowchart fordetection of cycling transcripts. (A) Populations of growth-synchronized wild-type L1 larvae were entrained for 5 d until adulthoodto 12 h:12 h LD cycles (500–1,000 lux) at a constant temperature of18uC, or for 4 d to 12 h:12 h temperature cycles (25u:15uC [WC]) in DD.RNA was collected every 4 h during the last entrainment and thesubsequent free-running days and analyzed via hybridization ofAffymetrix GeneChips (see Materials and Methods). L4 larvae weretransferred to FUDR-containing plates to inhibit embryonic develop-ment. (B) Normalized and standardized expression values from eachindependent time series experiment were prescreened with ANOVA toidentify transcripts that exhibit statistically significant changes inexpression over time. Appended datasets from each independent timeseries experiment were then examined to identify the 24-h spectralpower (F24) score of each transcript [87], and the significance wascalculated via comparison with a randomly permuted dataset. The 24-hautocorrelation (AC24 score) between time points that were 24 h apartwas determined by fitting the six time points collected during theentrainment day to the six time points collected during the free-running day. See Materials and Methods for additional details.doi:10.1371/journal.pbio.1000503.g001

Author Summary

Daily (circadian) rhythms in behavior and physiology alloworganisms to adapt to periodic cues such as light andtemperature associated with the rotation of the earth.Subsets of molecular components of the internal clock thatdrive these rhythms, as well as effector genes forbehavioral outputs, also exhibit rhythmic expression inmany organisms. While circadian rhythms in behavior havepreviously been described in the nematode Caenorhabditiselegans, no transcriptional rhythms or clock genes havebeen identified, leaving open the question of the nature ofthe clock in this organism. Here, we identify light- andtemperature-entrained cycling genes in C. elegans viagenome-wide transcriptional profiling. Transcripts show-ing circadian regulation (including expression with a 24-hperiod maintained upon removal of the entrainingstimulus) and temperature compensation were identified.Light and temperature appear to entrain independent setsof genes. We also identify large sets of light- ortemperature-driven genes. Mutations in a channel genepreviously implicated in sensory transduction in a small setof sensory neurons abolish entrainment of gene expres-sion by environmental signals. This work demonstrates thepresence of circadian transcriptional rhythms in C. elegans,and provides the foundation for future investigations intothe underlying mechanisms.

Circadian Transcripts in C. elegans


To identify putative circadian oscillatory transcripts with high

confidence, we performed a series of data analyses based on the

initial assumption that these rhythms exhibit approximately 24-h

periodicity (Figure 1B and Table S1; see Materials and Methods).

These algorithms identify cycling transcripts based on consonances

in their periods and phases while minimizing the contribution of

experimental variations in oscillation amplitude [46]. Similar

methods were used in a meta-analysis of circadian expression

profiling data from Drosophila to identify a set of light- and

temperature-entrained circadian transcripts [41,47]. To validate

the algorithms and filters applied by us, we reanalyzed a LD

dataset from Drosophila (S. K. and M. R., unpublished data) and

successfully identified all major core clock genes including per, clk,

tim, cry, and vri, whose expression is known to cycle under these

conditions (data not shown). These analyses allowed us to identify

transcripts that cycle with a 24-h period in both the light- and

temperature-regulated datasets, subsets of which continued to

cycle under constant conditions (see below).

Transcription Rhythms Are Driven, Entrained, andEnriched for 24-h Periods

Entrainment via light or temperature cycles may drive as well as

entrain transcriptional rhythms [47]. Stimulus-driven but clock-

independent transcripts are expected to cycle during entrainment

but not during free-running conditions, as observed in the first

category of cycling transcripts (LD only or WC only) (Figures 2A

and 3A). The second category included transcripts that cycled

during both entrainment and free-running conditions (LD/DD or

WC/CC) (Figures 2B and 3B), suggesting that expression of these

genes may be under circadian control [47]. We also identified a

third category of transcripts whose expression cycled during the

free-running but not during the entrainment conditions (DD only or

CC only) (Figures 2C and 3C). This category may include genes

whose expression is also under circadian control, but whose cycling

is suppressed or masked during entrainment conditions. A similar

category of transcripts was previously noted in genome-wide studies

of light-regulated transcriptional rhythms in Drosophila [36].

We investigated whether a 24-h circadian period is enriched in

our datasets by comparing the distribution of Fourier scores with

different periods with randomly permuted Fourier scores in

quantile–quantile (QQ) plots. All LD datasets (LD only, LD/

DD, and DD only) were significantly enriched for 24-h periods,

whereas distributions of 12-h, 18-h, and 36-h period data were

indistinguishable from those of randomly permuted data

(Figure 4A). Although 24-h periods were also similarly enriched

in the WC datasets (WC only, WC/CC, and CC only), we found

lower, but significant enrichment of 12-h and 36-h periods in the

WC-only and WC/CC datasets and in the CC-only datasets,

respectively (Figure 4B). These results indicate robust circadian

regulation of transcripts, with infradian and ultradian rhythms also

observable in the temperature-regulated datasets. This pattern is

similar to higher order harmonics of circadian gene expression

previously identified in mouse liver [48].

Temperature Cycles Drive the Expression of a Large Setof Genes Implicated in Metabolic Processes

We next further analyzed the light- and temperature-driven

transcripts (LD-only and WC-only datasets, respectively). The

Figure 2. Light-driven and -entrained transcripts. Phase-ordered heat maps showing cycling transcripts upon entrainment to LD cycles. (A)Transcripts that cycle in LD but not in DD. (B) Transcripts that cycle in both LD and DD. (C) Transcripts that cycle in DD but not in LD. Only transcriptsthat passed all applied filters and thresholds are shown (Table S1). The heat maps in (A) and (C) show appended data from three independent 1-dentrainment time series experiments and three independent 1-d free-running time series experiments, respectively. The heat map in (B) shows theaverage of three independent 2-d time series experiments. Data from two independent 1-d time series experiments performed in parallel in DD tocontrol for temperature fluctuations are also shown in (B). In all panels, columns represent experimental time points, and rows represent individualtranscript profiles. Rows are ordered according to the estimated peak phases of the transcript profiles across the appended or averaged datasets,indicated by the vertical bars to the right of each heat map. Expression values represented by the green-to-red color scale indicate up- or down-regulation relative to the experimental mean expression values indicated in black. Horizontal bars below the heat maps correspond to the LDentrainment protocols used in the time series experiments; white, black, and gray bars in all panels indicate the light, dark, and subjective lightphases, respectively.doi:10.1371/journal.pbio.1000503.g002



number of putative temperature-driven transcripts was signifi-

cantly higher than the number of light-driven transcripts: 1,817

WC-driven compared to 775 LD-driven transcripts (Figures 2A

and 3A). These comprise approximately 9% and 4% of all C.

elegans genes, respectively, underscoring the critical roles of

temperature and light in regulating C. elegans physiology.

We verified rhythmic expression of selected genes by perform-

ing quantitative reverse transcription PCR (qRT-PCR) analyses.

Each of the 12 examined genes from the WC-only dataset

exhibited significant cycling, with phases consistent with those

identified in the transcriptional profiling experiments (Figures 5A

and S1A). Expression was arrhythmic in the absence of imposed

temperature cycles (Figure S1B). These driven genes are predicted

to be implicated in diverse biological pathways (Figure 5B and

Table S2). Based on the biological process Gene Ontology (GO)

terms [49], categories of metabolism and electron transport were

markedly enriched in the WC-only dataset (Figure 5B and Table

S2). For instance, the expression of 18 genes predicted to encode

cytochrome P450 enzymes was driven by WC cycles, whereas only

two cytochrome P450 genes were present in the LD-driven dataset

(Table S2). This suggests that temperature, but not light,

modulates the expression of genes implicated in global regulation

of metabolism or physiology. Genes associated with the GO term

embryonic development were enriched in the LD-only-driven

dataset (Figure 5B and Table S2).

We ordered the transcripts in each dataset by the average phase

of peak expression (Figure 5C). We found that the peak phases

were around the middle of the thermo–light or the cryo–dark

phases. For LD transcripts, there were similar numbers of

transcripts at these two phases, whereas more WC transcripts

peaked during the cryophase than in the thermophase (Figure 5C),

similar to previous observations in Drosophila [47]. Together, these

results indicate that temperature and light cycles drive global

changes in gene expression in C. elegans.

Putative Circadian Transcripts Represent DiverseBiological Functions

In total, 406 and 286 transcripts were found to show oscillatory

expression during the DD and CC free-running conditions,

respectively (see Materials and Methods). The subset of these

transcripts that cycle both during the LD and WC entrainment

conditions and during free-running conditions may be driven as

well as entrained. We verified the cycling of selected transcripts by

performing qRT-PCR (Figures 6A and S2A). Their phases were

similar to those defined via expression profiling, and cycling was

similarly dependent on prior entrainment (Figure S2B). Following

entrainment, circadian genes sustain cycling in constant condi-

tions. albeit with a considerably diminished amplitude in some

systems over extended time periods. We therefore examined the

expression of two temperature-entrained genes (nlp-36 and

F23F12.3) through 2 d of free-running conditions. Both transcripts

continued to exhibit rhythmic transcription during the second

free-running day, with highly significant 24-h spectral power

values (Figure 6A).

Genes associated with the GO term categories of post-embryonic

morphogenesis and development were significantly enriched in both

circadian datasets, in contrast to the light- and temperature-driven

transcripts (Figure 6B and Table S3). We noted that genes

implicated in reproductive development, and to a lesser extent

locomotion, were enriched in the temperature-entrained dataset,

suggesting that processes such as mating, fertilization, egg-laying,

and locomotor behaviors may be under circadian control.

Figure 3. Temperature-driven and -entrained transcripts. Phase-ordered heat maps showing cycling transcripts upon entrainment totemperature cycles (25uC:15uC). (A) Transcripts that cycle in WC but not in CC. (B) Transcripts that cycle in both WC and CC. (C) Transcripts that cyclein CC but not in WC. Only transcripts that passed all applied filters and thresholds are shown (Table S1). The heat maps in (A) and (C) show appendeddata from three independent 1-d entrainment time series experiments and three independent 1-d free-running time series experiments, respectively.The heat map in (B) shows the average of three independent 2-d time series experiments. Data are presented as in Figure 2. Horizontal bars belowthe heat maps correspond to the WC entrainment protocols used in the time series experiments; red, blue, and light blue bars in all panels indicatethe warm, cold, and subjective warm phases, respectively.doi:10.1371/journal.pbio.1000503.g003



To determine the phase distribution of the light- and

temperature-entrained transcripts, we grouped the cycling tran-

scripts identified in the DD- and CC-entrained datasets in 4-h

clusters according to their phases of peak expression. The peak

phases of circadian transcripts in the light-entrained dataset

occurred at around the middle of the light and dark phases,

although with a relatively broad distribution (Figure 6C). The

phase distribution for temperature-entrained transcripts exhibited

a narrower peak in the middle of the thermophase, with another

smaller peak in the middle of the cryophase. Although these

distributions were not identical to those observed in the driven

data, there were some similarities: they both peaked in the middle

Figure 4. 24-h rhythms are enriched in the light- and temperature-entrained datasets. QQ plots of Fourier scores at four different periods(12, 18, 24, and 36 h) are shown from LD-entrained (A) and temperature-entrained (B) datasets subdivided into categories as indicated in Figures 2and 3. The Fourier scores of each transcript (with log2-transformed expression value .10) are graphed against the randomly permuted 95th quantileFourier scores. Enrichment of the experimental period is shown as an upward departure from the diagonal (red line), while depletion is shown as adownward departure.doi:10.1371/journal.pbio.1000503.g004

Figure 5. Analyses of light- and temperature-driven transcripts. (A) Comparison of qRT-PCR (gray) and microarray expression array (black)data for four arbitrarily selected WC-driven transcripts. Additional transcripts are shown in Figure S1. The probability of significance of circadiancycling (pF24) as compared to a randomized dataset was calculated by appending each independent microarray or qRT-PCR time course experiment.Bars below the graphs denote the entrainment protocols, with red and blue bars indicating the warm (25uC) and cold (15uC) phases, respectively.Data shown are an average of three biologically independent replicates per time point for the microarray data, and two biologically independentreplicates per time point for the qRT-PCR data. Data from two probe sets on the GeneChips for ceeh-1 are shown. (B) Functional enrichment of LD-and WC-driven transcripts. Transcripts are grouped by the Biological Process 3 GO term and analyzed for enrichment relative to all transcripts presenton the GeneChip (see Materials and Methods). p-values for enrichment for each group are indicated. A complete list is provided in Table S2. (C)Histogram showing the estimated peak phases of the temperature- or light-driven transcripts. The peak phases are organized into 4-h phase groups.doi:10.1371/journal.pbio.1000503.g005



of the light (warm) and dark (cold) phases and with similar

numbers of LD-entrained or -driven transcripts in the two peaks.

However, more WC-driven transcripts peaked in the cryophase

whereas more WC-entrained transcripts peaked in the thermo-

phase (compare Figures 5C and 6C).

The Periods of Putative Circadian Transcripts AreTemperature-Compensated, and Their Phases AreAltered by T-Cycles

A hallmark of circadian rhythms is temperature compensa-

tion, i.e., the circadian period is almost independent of

temperature [50,51]. To compare periods of two light-entrained

transcripts upon growth at 15uC and 25uC, we examined

transcript levels via qRT-PCR. The calculated periods and

significant periodicities for each transcript were nearly identical

at both temperatures (Figure 7A and Table S4), indicating

temperature compensation.

Another characteristic of circadian rhythms is the maintenance

of a phase relationship with the period (T) of the zeitgeber [52–55].

Typically, phases are delayed relative to those in 24-h T-cycles

upon entrainment to T-cycles shorter than 24 h, and, conversely,

phases are advanced when entrained to T-cycles longer than 24 h.

We entrained animals to either 11 h:11 h or 13 h:13 h WC cycles

and examined the phases of two candidate circadian transcripts by

qRT-PCR. The phases of both transcripts were delayed in

response to 11 h:11 h T-cycles and advanced in response to

13 h:13 h T-cycles (Figure 7B), further suggesting that these

transcripts are under circadian regulation.

Light–Dark and Temperature Cycles Entrain IndependentGene Sets

We next compared the two pairs of datasets driven and

entrained by light and temperature cycles, to determine the extent

to which the two zeitgebers regulate similar sets of genes. A direct

Figure 6. Analyses of light- and temperature-entrained circadian transcripts. (A) Comparison of qRT-PCR (gray) and microarray expressionarray (black) data of randomly selected LD- and WC-entrained transcripts. Additional transcripts are shown in Figure S2. Bars below the graphs denotethe entrainment protocols, with white, black, and gray bars indicating the light, dark, and subjective light phases, respectively, and with red, blue, andlight blue indicating the warm (25uC), cold (15uC), and subjective warm phases, respectively. Data shown are an average of three biologicallyindependent replicates per time point for the microarray data and two biologically independent replicates per time point for the qRT-PCR data. (B)Functional enrichment of light- and temperature-entrained circadian transcripts, as shown in Figure 5B. A complete list is provided in Table S3. (C)Histogram showing the estimated peak phases of the temperature- or light-entrained transcripts. The peak phases are organized into 4-h phasegroups.doi:10.1371/journal.pbio.1000503.g006



comparison of the light- and temperature-driven datasets identi-

fied 107 transcripts in common, representing 106 genes

(Figure 8A). In the entrained sets, there were only two genes in

common (Figure 8B). One of these genes is predicted to be a

pseudogene (Y102A5C.6), and the second gene (swd-3.3) is

predicted to encode a homolog of the WDR5 member of the

histone methyltransferase complex. Interestingly, this protein is

known to associate with mammalian PER1 and regulate its

functions [56].

Since the extent of overlap between the datasets is highly

dependent on the applied filters and thresholds, we also examined

the correlation between the F24 scores for light–dark and

temperature regulation for each transcript in the driven and

entrained datasets. We found strong negative correlation in both

cases (Figure 8C and 8D), further implying that light–dark and

temperature cycles regulate independent sets of genes.

We considered the possibility that the lack of overlap between

the entrained datasets may be due to the presence of additional

circadian transcripts in the driven datasets. The expression of these

transcripts may dampen quickly in constant conditions, precluding

their inclusion in the entrained datasets. These transcripts may be

distinguished from truly driven genes by being phase-shifted in

response to T-cycles that are longer or shorter than 24 h. To

address this hypothesis, we subjected seven arbitrarily selected

transcripts from the WC-only-driven datasets to 11 h:11 h or

13 h:13 h T-cycles. Although the phases of six of seven of these

transcripts did not exhibit the expected shifts, the phase of dpy-7

was advanced or delayed similarly to the examined entrained

transcripts (Figures 7C and S3). This observation suggests that the

driven datasets may include additional entrained genes, some of

which may be entrained by both light and temperature.

Clock Gene Homologs in C. elegans Do Not ExhibitRhythmic Expression in Whole Animal ProfilingExperiments

Although a subset of core clock genes, such as the doubletime

casein kinase gene, are expressed constitutively [57], genes such as

per or tim exhibit circadian transcription in Drosophila [23–26]. The

C. elegans genome is predicted to encode homologs of core clock

genes implicated in circadian regulation in Drosophila and

Figure 7. Temperature compensation and phase shifts of entrained transcripts. (A) Cycling of two light-entrained transcripts during thefirst 2 d of free-running conditions at 15uC (blue line) or 25uC (red line) quantified via qRT-PCR. Calculated periods are as follows: K11H12.6,23.560.40 h at 15uC and 23.9060.70 at 25uC; C33F10.4, 23.3560.25 h at 15uC and 23.4560.25 h at 25uC. Fourier scores and associated probabilitiesare shown in Table S4. The bars below the graphs denote the entrainment protocol, with black and gray bars indicating the dark and subjective lightphases, respectively. Data shown are from two biological replicates. (B and C) Phase shifts upon entrainment to different T-cycles. Shown is theexpression of two genes each from the WC-entrained (B) and WC-driven (C) datasets as quantified from GeneChip or qRT-PCR data. RNA wascollected on the fourth entrainment day. The expression of additional driven transcripts is shown in Figure S3. Data shown are the average of twotechnical replicates from one biological experiment.doi:10.1371/journal.pbio.1000503.g007



mammals (www.wormbase.org) [21], and animals mutant for the

per homolog lin-42 exhibit an increased circadian period in

locomotor behavior upon entrainment to light or temperature

cycles [19]. However, upon entrainment by either light or

temperature zeitgebers, no significant rhythmicity was found for

the expression of the lin-42 (per), tim-1 (tim), aha-1 (clk/cyc), or atf-2

(vri) genes (Table S5). We also did not observe changes in green

fluorescent protein (gfp) expression upon temperature entrainment of

wild-type animals transgenic for atf-2p::gfp and aha-1p::gfp fusion

genes (data not shown). It remains possible, however, that clock

gene homologs cycle in a small subset of cells or tissues in C. elegans,

and/or that these genes exhibit rhythmic expression at the post-

transcriptional level.

Cycling Expression of the nlp-36 Neuropeptide Gene CanBe Monitored In Vivo

To monitor the rhythmic expression of candidate entrained

genes in vivo in C. elegans, we established a real-time gfp-based

reporter system. Expression of the nlp-36 neuropeptide gene

exhibits strong temperature-entrained rhythmic expression

(Figure 6A and Table S3). We generated strains carrying stably

integrated transgenes in which the nlp-36 promoter drives

expression of the gfp reporter gene fused to sequences encoding

a rapidly turning over PEST domain [58]. The nlp-36p::gfp fusion

gene was expressed in the intestine and additional unidentified

cells in the head and tail in adult animals (Figure 9A). We

subjected transgenic animals to either cold/warm (CW) or WC

cycles and quantified GFP fluorescence levels in the intestine at

defined time points.

nlp-36p::gfp expression cycled robustly both during entrainment

and under free-running conditions, with the expected opposite

relative phases in response to CW or WC cycles (Figure 9B).

Moreover, cycling persisted for at least two subsequent days in

free-running conditions, albeit with dampened amplitude

(Figure 10A). Consistent with the absence of nlp-36 in the set of

light-entrained transcripts, we did not observe cycling of nlp-

36p::gfp with light entrainment (Figure S4).

To determine whether the phase of cycling nlp-36p::gfp

expression could be reset upon exposure to a different entraining

zeitgeber schedule, we entrained nlp-36p::gfp-expressing transgenic

animals to 12 h:12 h CW cycles for 4 d. The animals were then

shifted to 25uC conditions for 6 h, subjected to cold temperatures

(15uC) for 6 h, and returned to 25uC. Quantification of intestinal

gfp expression levels showed that administration of this tempera-

ture pulse resulted in a phase delay (Figure 9C), indicating that nlp-

36 cycling is regulated by a circadian clock.

Light and Temperature Entrainment of nlp-36 ExpressionRequires the tax-2 Cyclic Nucleotide-Gated Channel

Molecules required to transduce light zeitgeber information to

the clock have been well studied [59–64], although the molecular

mechanisms and circuits that transduce temperature signals are less

well understood. In C. elegans, the tax-2 subunit of a cyclic nucleotide-

gated channel has been shown to be required for responses to

temperature as well as to short-wavelength visible and ultraviolet

light [42–44,65,66]. We found that temperature-entrained rhyth-

mic expression of nlp-36p::gfp expression in the intestine was

significantly affected both during entrainment and free-running

conditions in tax-2 mutants (Figure 10B). Similarly, light-entrained

circadian transcription of three examined genes, as measured by

qRT-PCR, was also abolished in tax-2 mutants (Figures 10C),

indicating that tax-2 is required for transduction of both light and

temperature zeitgebers to the circadian clock(s). However, light-

entrained cycling was unaffected in animals mutant for the

transmembrane LITE-1 light receptor protein also shown to be

required for light responses in C. elegans (Figure 10D) [42,44,66].

Figure 8. Temperature and light entrain and drive the expression of distinct genes. (A and B) Overlap between light- and temperature-driven cycling transcripts (A), and light- and temperature-entrained cycling transcripts (B). (C and D) Scatter plots of the F24 scores of each transcript inthe driven (C) or the entrained (D) datasets. F24 scores for light–dark and temperature regulation are shown in black and gray, respectively.doi:10.1371/journal.pbio.1000503.g008



tax-2 is expressed in several sensory neuron types, including AFD

thermosensory neurons, which is the primary thermosensory neuron

type in C. elegans [67,68]. We found that nlp-36p::gfp expression

continued to cycle, albeit with markedly reduced amplitude, in

animals in which the AFD neurons had been genetically ablated

(Figure 10A; gift of Miriam Goodman, Stanford University). This

result suggests that the AFD neurons are not the sole source of

temperature information for the clock and that one or more tax-2-

expressing sensory neurons are required to sense and transduce

environmental temperature information to entrain rhythms.

Discussion

In the present study, we report the first identification, to our

knowledge, of circadian-regulated transcripts in C. elegans. These

transcripts are entrained by LD or WC cycles and continue to oscillate

during free-running conditions with temperature-compensated peri-

ods and phases that are dependent on entraining conditions. Both

light and temperature can act as zeitgebers, and they drive as well as

entrain transcripts at the genome-wide level. Genome-wide circadian

expression is regulated by environmental information received by

a small set of sensory neuron(s), suggesting that, similar to observations

in vertebrates, light and temperature information may act cell-

nonautonomously to entrain the clock(s) in C. elegans. Our findings

also imply that C. elegans may utilize a timekeeping mechanism

that is distinct from that described previously in other animals.

In C. elegans, as well as in Drosophila, temperature cycles drive the

expression of a larger set of genes than do light cycles [41,47].

Genes implicated in metabolism appear to be enriched in the

temperature-driven transcript sets, whereas genes implicated in

circadian behaviors such as locomotion and reproduction are

enriched in the entrained datasets in both organisms ([47] and this

study). However, there is a surprising lack of overlap between the

temperature- and light-entrained datasets in C. elegans. In contrast,

in Drosophila, light and temperature cooperatively entrain a single

transcriptional clock [47,69,70].

What does this almost complete lack of overlap between the two C.

elegans entrained datasets imply? At one extreme, light and

temperature might act via different mechanisms to entrain two

distinct clocks. Multiple clocks within a single organism are not

unprecedented [4], although there is no evidence for a similar

situation in metazoans. On the contrary, Drosophila has only a single

clock that runs in multiple cells and tissues [47,69]. Similarly, C. elegans

may have a single clock that is entrained by light or temperature in

different tissues. In this scenario, the different locations may have

completely distinct sets of output genes, accounting for the

nonoverlapping datasets. However, in this case, cycling clock mRNAs

are expected to be shared in common between the two sets since in all

eukaryotic circadian systems, from plants to mammals, many clock

mRNAs undergo circadian oscillations at the transcriptional level

[23,24,71,72]. Since it is unlikely that a clock based on transcriptional

feedback loops would have only one cycling mRNA (e.g., the swd-3.3

gene), C. elegans may have a novel clock mechanism. Consistent with

this hypothesis, homologs of animal clock genes do not cycle in C.

elegans. Recent analysis has suggested that the ascidian Ciona intestinalis

also has a divergent circadian clock, further suggesting multiple

evolutionary origins of the animal circadian mechanism [73].

However, it remains possible that cycling clock mRNAs were not

identified in our genome-wide studies because of masking by

noncycling expression in other tissue types, or that clock proteins

exhibit functional rhythmicity in C. elegans via post-translational

modifications [74–76].

An alternative possibility, consistent with the presence of a single

clock, is that cycling clock mRNAs as well as common output genes

are present in the two driven datasets. These datasets have a

statistically significant overlap, which is expected for clock and clock-

regulated mRNAs. However, these transcripts do not appear to

maintain cycling in free-running conditions. Although free-running

rhythms are considered to be the sine qua non for circadian genes,

some bona fide circadian rhythms damp quickly in constant

conditions. For example, Drosophila peripheral rhythms damp rapidly

in the absence of a light–dark cycle, including transcriptional rhythms

of the clock genes themselves [77,78]. Consistent with this scenario, we

identified one of seven examined transcripts in the WC-driven datasets

with characteristics of an entrained rather than a strictly driven

transcript. The number of entrained transcripts is, therefore, likely to

be larger, and both clock mRNAs and shared output genes may be

present within the set of entrained genes whose expression is severely

dampened under constant conditions. Further molecular and genetic

analyses will allow us to distinguish among the above possibilities.

What are the behavioral consequences of circadian rhythmicity

in C. elegans? Although locomotion has been reported to be under

circadian control and genes implicated in locomotor behaviors are

enriched in the WC-entrained dataset, this behavioral output does

not appear to be particularly robust, and exhibits marked animal-

to-animal variability [16,19]. Locomotor behaviors in C. elegans are

complex and can be dissected into multiple behavioral compo-

nents [79–83]. Reported analyses of circadian locomotor behavior

relied largely on quantification of gross overall movement [16,18],

suggesting that detailed analyses of the underlying components

may reveal more robust clock control. Since reproduction-related

genes are also enriched in the entrained datasets, additional

biologically relevant behaviors such as egg-laying or mating may

also be regulated in a circadian manner in C. elegans. Identification

of circadian transcriptional rhythms in C. elegans now provides the

necessary reagents to characterize the molecular identity, the

neuronal circuits, and the behavioral consequences of clock

function in this important model organism.

Materials and Methods

C. elegans Strains and Culture ConditionsThe wild-type strain used was C. elegans variety Bristol, strain

N2. Animals were cultured on Escherichia coli HB101. Mutant

strains used were the following: PR671 tax-2(p671) [65], KG1180

lite-1(ce314) [66], and GN112 (pgIs2) (gift of M. Goodman,

Stanford University). The AFD neurons are ablated in the

GN112 strain via expression of reconstituted caspases under the

AFD-specific gcy-8 promoter [84,85]. Double mutant strains were

constructed using standard methods.

Figure 9. Expression of an nlp-36p::gfp fusion gene is entrained by temperature cycles. (A) gfp expression in transgenic animals carryingstably integrated copies of an nlp-36p::gfp fusion gene during temperature entrainment (CW) and free-running (WW) conditions. Adult animals wereexamined under 1006magnification. (B) Quantification of intestinal nlp-36p::gfp fluorescence intensity (in arbitrary units [A.U.]) at the indicated timepoints during entrainment or free-running days. Wild-type transgenic animals were entrained to either WC/CC (gray bars) or CW/WW (red bars) cycles.Error bars indicate the s.e.m. Data shown are from two independent experiments with 15–20 animals at each time point. (C) nlp-36p::gfp-expressingtransgenic animals were subjected to CW entrainment cycles and moved to WW conditions. After 6 h at WW, a subset of animals were subjected to apulse of 15uC temperature for 6 h and returned to WW conditions. Intestinal fluorescence intensities in animals subjected or not subjected to the coldpulse are indicated in blue and gray, respectively. Error bars indicate the standard deviation. n = 15–20 animals each at each time point.doi:10.1371/journal.pbio.1000503.g009



Entrainment MethodsGrowth-synchronized populations of wild-type late L1 larvae

were transferred to plates seeded with bacteria. Two populations

of animals were entrained to opposing light–dark or temperature

cycles in two different incubators: 12 h at 25uC followed by 12 h

at 15uC (WC), or to 12 h of light followed by 12 h of dark (LD).

After reaching adulthood, the two populations of animals were

transferred to the same incubator and allowed to free-run at

constant 15uC (CC) or in constant darkness (DD). The time point

0 h indicates lights on or the start of the warm phase, 12 h

Figure 10. Rhythmic expression of nlp-36p::gfp is abolished in tax-2 mutants. (A) Quantification of intestinal nlp-36p::gfp expression levels inwild-type (gray bars) or AFD-killed animals (red bars) at defined time points during entrainment (CW) followed by 3 d of free-running (WW)conditions. Error bars indicate the s.e.m. Data shown are from two independent experiments of 20 animals each. (B) Fluorescence intensities in wild-type animals (gray bars) or tax-2 mutant animals (red bars) carrying the nlp-36p::gfp transgene entrained to temperature cycles (CW/WW). Error barsindicate the s.e.m. Data shown are from two independent experiments. (C) Cycling of selected light-entrained transcripts in wild-type animals (gray)and in tax-2(p671) mutants (red) quantified via qRT-PCR. The probabilities of circadian cycling (pF24) by appending each independent qPCR timecourse experiment are indicated. (D) Cycling of selected light-entrained transcripts in wild-type animals (gray) and in lite-1(ce314) mutants (green)quantified via qRT-PCR. The probabilities of circadian cycling (pF24) are indicated. The bars below the graphs in (C) and (D) denote the entrainmentprotocol, with white and black bars indicating the light and dark phases, respectively. Data shown are from two independent biological replicates pertime point. Error bars indicate the s.e.m. n = 15–20 animals each.doi:10.1371/journal.pbio.1000503.g010



indicates lights off or the start of the cold phase. Temperature

cycling experiments were conducted in DD in temperature

programmable incubators (Tritech Research). Light–dark cycling

experiments were conducted at 18uC in programmable incubators

(Precision Scientific; cold white light [light source F20T12/CW] at

about 500–1,000 lux). To inhibit progeny production during

entrainment, L4 larvae were quickly transferred to plates

containing bacteria and 25 mM FUDR. Adults were harvested

and washed in cold 16M9 buffer (,15 min) every 4 h during the

last day of entrainment and subsequent free-running days.

Microarray HybridizationTrizol Reagent (Invitrogen) was added to harvested worm

samples and pellets were immediately frozen in liquid nitrogen.

Extracted total RNA was treated with DNAase (New England

Biolabs) as described [86]. cDNA synthesis was performed using

T7-oligo(dT) primer and Superscript II (Affymetrix). Biotin-

labeled cRNA was generated using the GeneChip IVT labeling

kit (Affymetrix) and following the methods described in the

Affymetrix GeneChip manual. Hybridization, washing, staining,

and scanning of the cRNAs to the Affymetrix C. elegans Genome

Arrays were performed as recommended by the manufacturer.

Microarray Data AnalysisExpression, normalization, and standardization. The

Affymetrix software was used to scan and generate .DAT and

.CEL files of microarrays. Raw data can be accessed in Gene

Expression Omnibus (www.ncbi.nlm.nih.gov/geo/; accession

number GSE23528). The GeneChip Robust Multiarray

Averaging method in the R v2.7.1 software package was used to

derive log2 expression values for each probe set from Affymetrix-

generated .CEL files. Expression values were normalized and

standardized as described [87]. Briefly, for each time point in a

time series experiment, the expression level relative to the mean (in

log2 expression values) over that experiment was calculated. This

normalization was performed separately for each independent

time series experiment. Arrays in the independent time course

experiments were standardized by setting the mean expression of

each array to 0 and the variance to 1 [87]. Transcripts with low

average signal intensities were excluded from further analyses.

ANOVA prescreening. Normalized and standardized

expression values from each independent time series experiment

were prescreened with ANOVA using a custom-written MATLAB

code [87] (generously provided by K. Keegan). For each transcript

on the arrays, a single-factor ANOVA was performed across all six

time points during the entrainment or the free-running days,

where each time point is a group and each array is an individual

within the group. An ANOVA p-value of less than 0.05 was

required for a transcript to pass prescreening.

Fourier analysis. Circadian oscillatory transcripts with 24-h

periods were identified by Fourier transformation. Normalized

and standardized data (see above) for each independent time series

experiment were appended, and the 24-h spectral power (F24) was

determined for each transcript. The spectral power score and

significance of this power (power p-value) generated via

comparison with a randomly permuted dataset (1,000

permutations) for all analyzed periods were obtained using a

custom-written MATLAB code [87] (provided by K. Keegan). We

elected to use a single ordering of the days in the appended profile,

since these analyses (with the exception of the AC24 score, see

below) are insensitive to the order of the days in the appended time

series experiments. Transcripts that exhibited pF24-values of less

than 0.02 were analyzed further.

Autocorrelation. An autocorrelation (AC24) method was

used to measure the correlation between time points that are

24 h apart by fitting the six time points collected during the

entrainment day to the six time points collected during the free-

running day. A previously described R code [46] was used to

calculate AC24 scores. Transcripts that passed this analysis were

required to have an average AC24 score determined from three

independent 2-d time series experiments of greater than zero.

False discovery rate analysis. To estimate the false positive

discovery rate, the expression values of time series experiments for

all datasets were randomly permuted (see Table S1). These

randomly permuted data were used to perform ANOVA and F24

analysis as described above. The percentage of false discovery rate

in Table S1 represents the percentage of unpermuted transcripts

that were identified in the randomly permuted dataset.

Fold change. Transcripts exhibiting an average fold change

of greater than 1.5 (log2-transformed expression values) between

their highest and lowest expression values among the six time

points of a time series experiment were selected.

QQ PlotsQQ plots were generated to determine whether particular

periods are enriched in the microarray datasets [41]. Briefly, 1,000

permutations of the ordering of time points for all transcripts

within each appended time series experiment (LD only, DD only,

LD/DD, WC only, CC only, and WC/CC datasets) were

conducted, and the permuted Fourier score for the resulting time

series experiments was calculated. The 1,000 permuted Fourier

scores were then divided into a number of quantiles equal to the

number represented in the unpermuted dataset. The distributions

of Fourier scores found in unpermuted data and the permuted

data quantiles were then compared and shown in QQ plots.

Gene Functional Annotation AnalysisCandidate-driven and circadian-entrained transcripts were

grouped by their Biological Process 3 GO term and analyzed for

enrichment relative to all transcripts present on the GeneChip

using functional annotation tools in DAVID (http://david.abcc.

ncifcrf.gov/).

qRT-PCRcDNA synthesis was performed from total RNA using T7-

oligo(dT) primer and Superscript III (Invitrogen). Real-time qRT-

PCR analysis was performed using a Corbett Research Rotor-

Gene 3000 thermal cycler. Normalization was carried out using

the act-3 actin or the ard-1 short-chain alcohol dehydrogenase

genes (expression levels of these genes do not appear to cycle).

Primer sequences are available upon request. pF24-values were

assessed as described above.

In Vivo Monitoring of gfp-pest ExpressionAbout 3.5-kb upstream sequences of the nlp-36 predicted

neuropeptide gene were fused to gfp-encoding sequences that

included a PEST domain [58]. Multiple copies of the nlp-36p::gfp-

pest transgene were stably integrated into the genome of wild-type

animals together with the unc-122p::dsRed marker to generate the

PY7644 (oyIs80) strain. To obtain a growth-synchronized popu-

lation of L1 larvae, ten adult animals were transferred to seeded

plates and were allowed to lay approximately 50 eggs. Wild-type

animals, tax-2 mutants, and AFD-killed L1 larvae carrying the

oyIs80 transgene were entrained for 4 d to temperature cycles

(12 h:12 h) under well-fed conditions, and intestinal nlp-36p::gfp-

pest fluorescence intensity was determined at specific time points



during entrainment and free-running days. No FUDR was added

during the entrainment protocol. Images of 15–20 adult animals

were acquired at 1006 magnification using a compound

microscope (Zeiss) equipped with epifluorescence and a CCD

camera (Hamamatsu Photonics) at each time point. Levels of

intestinal expression were quantified in arbitrary units using Image

J (National Institutes of Health).

Supporting Information

Figure S1 Analysis of temperature-driven transcripts.(A) Comparison of qRT-PCR (gray) and microarray expression

array (black) data of randomly selected WC-driven transcripts.

The probability of significance of circadian cycling (pF24) as

compared to a randomized dataset was calculated by appending

each independent dataset. There are two probe sets each for acs-2

and cyp-35C1 on the GeneChips. Bars below the graphs denote the

entrainment protocols, with red and blue bars indicating the warm

(25uC) and cold (15uC) phases, respectively. (B) qRT-PCR data of

temperature-driven transcripts under constant conditions (15uC).

Data shown are an average of three biologically independent

replicates per time point for the microarray data and two

biologically independent replicates per time point for the qRT-

PCR data.

Found at: doi:10.1371/journal.pbio.1000503.s001 (0.70 MB TIF)

Figure S2 Analysis of light- and temperature-entrainedtranscripts. (A) Comparison of qRT-PCR (gray) and microarray

expression array (black) data of arbitrarily selected light- and

temperature-entrained transcripts. The probability of significance of

circadian cycling (pF24) as compared to a randomized dataset was

calculated by appending each independent microarray or qRT-

PCR time course experiment. Bars below the graphs denote the

entrainment protocols, with white, black, and gray bars indicating

the light, dark, and subjective light phases, respectively, and with

red, blue, and light blue bars indicating the warm (25uC), cold

(15uC), and subjective warm phases, respectively. Data shown are

an average of three biologically independent replicates per time

point for the microarray data, and two biologically independent

replicates per time point for the qRT-PCR data. (B) qRT-PCR data

of light- and temperature-entrained transcripts under constant

conditions. Data shown are an average of two biologically

independent replicates per time point for the microarray data,

and two biologically independent replicates per time point for the

qRT-PCR data, with the exception of K11H12.6 and C33F10.4, for

which data from one experiment were analyzed.


Figure S3 Phases of transcripts from the WC-drivendatasets. Animals were entrained to the indicated T-cycles for

3 d and RNA was collected on the fourth day. qRT-PCR data for

each time point are the average of two technical replicates from

one biological experiment.


Figure S4 nlp-36p::gfp expression does not cycle uponlight entrainment. Fluorescence intensities in wild-type animals

(gray bars) or tax-2 mutant animals (red bars) carrying the nlp-

36p::gfp transgene entrained to light cycles (LD/DD). Error bars

indicate the standard error of the mean (s.e.m). Data shown are

from two independent experiments.


Table S1 False discovery rate analysis of datasets.

Found at: doi:10.1371/journal.pbio.1000503.s005 (0.11 MB

DOC)

Table S2 GO categories and lists of all light- andtemperature-driven transcripts.

Found at: doi:10.1371/journal.pbio.1000503.s006 (1.18 MB XLS)

Table S3 GO categories and lists of all light- andtemperature-entrained transcripts.

Found at: doi:10.1371/journal.pbio.1000503.s007 (0.47 MB XLS)

Table S4 Fourier analyses of periods at differenttemperatures.


DOC)

Table S5 Transcripts of C. elegans clock genes do notoscillate in a circadian manner.


DOC)

Acknowledgments

We are grateful to Felix Naef, Herman Wijnen, and Kevin Keegan for help

with data analyses, Rinho Kim for technical assistance, and Miriam

Goodman for reagents.

Author Contributions

The author(s) have made the following declarations about their

contributions: Conceived and designed the experiments: AMvdL MR

PS. Performed the experiments: AMvdL SK SW. Analyzed the data:

AMvdL MB. Contributed reagents/materials/analysis tools: MB JR.

Wrote the paper: AMvdL MR PS.

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